In the past year we have been successful in obtaining a monoclonal antibody that recognizes the plasminogen activator (PA) from Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF). The antibody has been selected for its ability not only to bind to PA but also to inhibit its catalytic activity. It is a highly specific antibody in tht it does not bind to or inhibit human urokinase or the PA activity present in cultures of human tumor cells, but does inhbit (at l-10 mu g/ml) the PA activity released by cultures of RSVCEF. The anti-catalytic activity of the antibody allows us now to directly test the role of PA in the behavior of cultures of RSVCEF. We have recently developed a biochemical assay for the degradation and invasive behavior of RSVCEF. The assay involves the degradation of the extracellular matrix (ECM) produced by normal CEF when cultures of RSVCEF are placed on radiolabeled ECM. The assay system allows us to test for the involvement of specific proteases and also for the requirement of cell-ECM contact. We plan to test for the direct role of PA in ECM degradation by conducting degradation studies in the presence or absence of our antibody. We plan to isolate a monoclonal antibody that binds to PA but does not inhibit its activity and this will serve as a useful control. In addition to using the antibody to test for PA's role in cellular behavior, we have now linked the antibody to solid support and are using it as an immunoaffinity ligand for purifying PA. In tandem with standard chromatography procedures the immunoaffinity column has yielded a homogeneous preparation of PA. This methodology will provide us with sufficient quantities of PA (0.1 to 1.0 mg), heretofore not available, to characterize the enzyme biochemically and use it to search for other relevant substrates. Our preliminary studies indicate that PA (in the absence of plasminogen) can cleave a homogeneous preparation of chick cellular fibronectin. These studies will be important in defining the exact role of the enzyme in certain aspects of cellular behavior including growth arrest and cell migration. We have recently established a tumor cell metastasis assay using the chick embryo. The system involves implanting human tumor cells on the chorioallantoic membrane (CAM) of the embryo and subsequently detecting human cells in peripheral organs of the embryo. There is a large body of indirect evidence suggesting a role for proteolytic enzymes in tumor cell invasion and metastasis implicating both tumor cell enzymes and host enzymes. Having procured an anti-catalytic antibody to human PA (urokinase) and now having an anti-catalytic antibody to chick PA, we will now be able to examine and dissect out the respective roles of tumor cell PA and host cell PA in the chick embryo metastasis system. (A)